This proposal is focused upon the intracellular amastigote stage of Leishmania mexicana complex parasites and the immune/inflammatory interactions occurring within the vaccinated host. Vaccination with the P-8 proteoglycolipid complex, which is expressed in the amastigote stage, confers resistance to infection. Studies indicate that CD8+ and CD4+ T cells are critical for resistance/protection against infection in P-8 immunized mice. Recent studies indicate that protection is dependent upon IFNg, perforin-mediated mechanisms and CD1d presentation. In addition, the associated P-8 glycolipids (1-4) directly affect macrophage function (cytokine production and parasite uptake). Consequently, this proposal is focused on elucidating the immune protective mechanisms induced by P-8 vaccination and on the biological roles (structure-function) of the P-8 glycolipids. Specifically, the aims are: 1. Determine the mechanisms by which CDld presentation/NKT activation contribute to the protection against Leishmania infection induced by P-8 vaccination. Using the murine model, the role of CDld presentation and NKT cells in the induction and effector phases of the protective immune response induced by P-8 vaccination will be determined. Further, CD1d-P-8 antigen presentation (kinetics, role of processing) by antigen-pulsed and infected macrophages and dendritic cells will be examined. 2. Investigate the biological function and biochemical characterization of the P-8 amastigote antigen. The P-8 antigen has been demonstrated to be a proteoglycolipid complex. The P-8 glycolipids affect macro-phage morphology and elicit cytokine/chemokine production; these glycolipids appear to be distinct from previously described leishmanial glycolipids. The biochemical structure of the P-8 glycolipids and the receptor (TLR; non-TLR) involved in macrophage activation will be determined. The CD1d -P-8 glycolipid(s) interaction will be investigated and KB determined using competitive binding and/or BIAcor methods. As the P-8cl serine proteinase complex appears to be antigenically important for protection and CD4 T cells contribute to protection, the gene encoding the p56 serine proteinase will be cloned and characterized.